We are investigating the mechanism of transport of D-Mannitol through the cytoplasmic membrane of Escherichia coli. The protein responsible for this process, the D-mannitol specific Enzyme II of the phosphoenolpyruvate: sugar phosphotransferase system (PTS), has been purified to homogeneity and some of its properties have been determined. This integral membrane permease catalyzes the concomitant vectorial transport and phosphorylation of D-mannitol in a highly specific manner. Thus, it offers the distinct advantage of possessing an enzymic activity coupled to a transport function and can be studied using classical enzymological methods. Specifically, we are continuing to investigate: 1) The orientation of the mannitol Enzyme II polypeptide in the membrane using proteolytic enzymes, specific antibodies and chemical modification. 2) The mechanism of the transport and phosphorylation reactions using inhibitors coupled with catalytic and binding studies. 3) The amino acid sequence of the protein, particularly as this relates to its function and intramembrane topography.